In the central nervous system (CNS) the transmission of stimuli takes place by the interaction of a neurotransmitter, which is sent out by a neuron, with a neuroreceptor.
L-glutamic acid, the most commonly occurring neurotransmitter in the CNS, plays a critical role in a large number of physiological processes. The glutamate-dependent stimulus receptors are divided into two main groups. The first main group forms ligand-controlled ion channels. The metabotropic glutamate receptors (mGluR) form the second main group and, furthermore, belong to the family of G-protein-coupled receptors.
At present, eight different members of these mGluR are known and of these some even have sub-types. On the basis of structural parameters, the different influences on the synthesis of secondary metabolites and the different affinity to low-molecular weight chemical compounds, these eight receptors can be subdivided into three sub-groups: mGluR1 and mGluR5 belong to group I, mGluR2 and mGluR3 belong to group II and mGluR4, mGluR6, mGluR7 and mGluR8 belong to group III.
Ligands of metabotropic glutamate receptors belonging to the group II can be used for the treatment or prevention of acute and/or chronic neurological disorders such as psychosis, schizophrenia, Alzheimer""s disease, cognitive disorders and memory deficits.
The present invention relates to compounds of the general formula I: 
These compounds have been discovered to act as metabotropic glutamate receptor antagonists and accordingly are usefull for the treatment of a range of neurological disorders, including psychosis, schizophrenia, Alzheimer""s and other cognitive and memory disorders.
Objects of the present invention are compounds of formula I and their pharmaceutically acceptable salts per se and as pharmaceutically active substances, their manufacture, medicaments based on one or more compounds in accordance with the invention and their production, as well as the use of the compounds in accordance with the invention in the control or prevention of neurological disorders, and, respectively, for the production of corresponding medicaments.
The present invention relates to compounds Of formula I 
wherein
X is a single bond or an ethynediyl group, wherein,
in case X is a single bond,
R1 is hydrogen; halogen; nitro; lower alkyl; halo-lower alkyl; alkoxycarbonyl; lower cycloalkyl, optionally substituted with oxygen; benzoyl, optionally substituted with lower alkyl, halo-lower alkyl, or halogen; phenyl, optionally substituted with halogen, hydroxy, lower alkyl, halo-lower alkyl, lower cycloalkyl, or lower alkoxy, halo-lower alkoxy, or cyano; styrenyl; phenylethyl; naphthyl; biphenyl; benzofuranyl; or 5 or 6 membered heterocyclic ring, optionally substituted with oxo, benzyloxy, benzoyl, methanesulfonyl, benzenesulfonyl, or acetyl;
in case X is an ethynediyl group,
R1 is hydrogen; lower alkyl, optionally substituted with hydroxy; halo-lower alkyl; lower cycloalkyl, optionally substituted with hydroxy, lower alkyl, halo-lower alkyl, lower alkoxy, halo-lower alkoxy, or halogen; lower cycloalkenyl, optionally substituted with lower alkyl, halo-lower alkyl, lower alkoxy, halo-lower alkoxy, halogen, or oxo; lower alkenyl; phenyl, optionally substituted with halogen, lower alkyl, halo-lower alkyl, lower cycloalkyl, lower alkoxy, halo; 5 or 6 membered heterocyclic ring, optionally substituted with lower alkyl, halogen, oxo, benzyloxy, benzoyl, methanesulfonyl, benzenesulfonyl, acetyl, pivaloyl, tert. butoxycarbonyl, or tert. butylcarbonyl; or benzofuranyl;
R3 is phenyl; pyridine; thiophenyl or thiazolyl, which are optionally substituted with halogen, cyano, nitro, azido, hydroxy, carboxy, morpholine-4-carbonyl, carbamoyl, thiocarbamoyl, N-hydroxycarbamoyl, trimethylsilyl-ethynyl, or from lower alkyl, lower alkynyl, lower alkoxy, halo-lower alkyl, 4-lower alkyl-piperazine-1-carbonyl, lower alkylaminocarbonyl, which are optionally substituted by amino, lower alkylamino, acylamino, oxo, hydroxy; lower alkoxy, lower alkylthio, or carboxy which is optionally esterified or amidated; or a 5-membered aromatic heterocycle which is optionally substituted by amino, lower alkylamino, acylamino, oxo, hydroxy, lower alkoxy, lower alkylthio, carboxy which is optionally esterified with lower alkyl or amidated with lower alkylamino which is eventually substituted by hydroxy, or lower alkyl which is optionally substituted by halogen, hydroxy, amino, lower alkylamino, acylamino, or amidino, which is optionally substituted by lower alkyl, xe2x80x94C(NRRxe2x80x2)xe2x95x90NRxe2x80x3 (where R, Rxe2x80x2 and Rxe2x80x3 are hydrogen or lower alkyl), hydroxy, lower alkoxy, lower alkylthio, acyloxy, lower alkylsulfinyl, lower alkylsulfonyl, lower alkoxy-lower alkylsulfanyl, lower alkylsulfanyl, cycloalkylsulfinyl, cycloalkylsulfonyl, hydroxyimino, or lower alkoxyimino, which is optionally esterified or amidated, lower alkenyl, oxo, cyano, carbamoyloxy, or sulfamoyl which is optionally substituted by lower alkyl,
with the proviso that, if X is a single bond and R3 is pyridinyl, R1 is not hydrogen, or methyl;
and their pharmaceutically acceptable acid addition salts.
It has surprisingly been found that the compounds of formula I are metabotropic glutamate receptor antagonists. Compounds of formula I are distinguished by valuable therapeutic properties.
The compounds of the present invention can be used for the treatment or prevention of acute and/or chronic neurological disorders such as psychosis, schizophrenia, Alzheimer""s disease, cognitive disorders and memory deficits.
Other treatable indications in this connection are restricted brain function caused by bypass operations or transplants, poor blood supply to the brain, spinal cord injuries, head injuries, hypoxia caused by pregnancy, cardiac arrest and hypoglycaemia. Further treatable indications are chronic and acute pain, Huntington""s chorea, amyotrophic lateral sclerosis (ALS), dementia caused by AIDS, eye injuries, retinopathy, idiopathic parkinsonism or parkinsonism caused by medicaments as well as conditions which lead to glutamate-deficiency functions, such as e.g. muscle spasms, convulsions, migraine, urinary incontinence, nicotine addiction, opiate addiction, anxiety, vomiting, dyskinesia and depressions.
Objects of the present invention are compounds of formula I and their pharmaceutically acceptable salts per se and as pharmaceutically active substances, their manufacture, medicaments based on one or more compounds in accordance with the invention and their production, as well as the use of the compounds in accordance with the invention in the control or prevention of illnesses of the aforementioned kind, and, respectively, for the production of corresponding medicaments.
Preferred compounds of formula I are those in which R3 is phenyl substituted in meta position by cyano; halogen; or imidazolyl, which is optionally substituted by lower alkyl; or 1,3-thiazolyl which is optionally substituted by hydroxy-lower alkyl, carboxy, or xe2x80x94COxe2x80x94NHxe2x80x94(CH2)2OH; 1,3-oxazolyl; 1,2,3-triazolyl; 1,2,4-triazolyl which is optionally substituted with lower alkyl; tetrazolyl; or isoxazolyl, which is optionally substituted by lower alkyl;
The following are examples of such compounds:
3-(4-Oxo-7-phenylethynyl-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-benzonitrile;
4-(3-Chloro-phenyl)-8-phenylethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
4-(3-Imidazol-1-yl-phenyl)-8-phenylethynyl-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
3-[7-(4-Fluoro-phenyl)-4-oxo-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl]-benzonitrile;
8-(4-Fluoro-phenylethynyl)-4-(3-imidazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-phenylethynyl)-4-(3-[1,2,4]triazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-phenyl)-4-[3-(4-methyl-imidazol-1-yl)-phenyl]-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-2-methyl-phenyl)-4-(3-imidazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-2-hydroxy-phenyl)-4-(3-imidazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-phenylethynyl)-4-(3-tetrazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(2-Fluoro-phenyl)-4-(3-[1,2,3]triazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(4-Fluoro-phenyl)-4-[3-(3-methyl-isoxazol-5-yl)-phenyl]-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(2,4-Difluoro-phenyl)-4-(3-[1,2,3]triazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(2,3-Difluoro-phenyl)-4-(3-[1,2,3]triazol-1-yl-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
8-(2-Fluoro-phenyl)-4-[3-(4-hydroxymethyl-thiazol-2-yl)-phenyl]-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
2-{3-[7-(2-Fluoro-phenyl)-4-oxo-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl]-phenyl}-thiazole-4-carboxylic acid;
2-{3-[7-(4-Fluoro-phenyl)-4-oxo-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl]-phenyl}-4-methyl-thiazole-5-carboxylic acid (2-hydroxy-ethyl)-amide; and
4-[3-(4,5-Dimethyl-4H-[1,2,4]triazol-3-yl)-phenyl]-8-(4-fluoro-phenyl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
Compounds of formula I, in which R3 is thiophenyl, preferably tiophen-2-yl, optionally substituted with halogen, cyano; or pyridinyl, preferably pyridin-4-yl, optionally substituted, preferably in the 2-position, with halogen, or cyano; are also preferred.
The following are examples of such compounds:
8-(4-Fluoro-phenylethynyl)-4-(2-imidazol-1-yl-pyridin-4-yl)-1,3-dihydro-benzo[b][1,4]diazepin-2-one;
2-[7-(4-Fluoro-phenyl)-4-oxo-4,5-dihydro-1H-benzo[b][1,4]diazepin-2-yl]-thiophene-3-carbonitrile;
4-(4-Oxo-7-phenylethynyl-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-pyridine-2-carbonitrile; and
4-[7-(2,4-Difluoro-phenyl)-4-oxo-4,5-dihydro-3H-benzo[b][1,4]diazepin-2yl]-pyridine-2-carbonitrile.
All tautomeric forms of the compounds of invention are also embraced herewith.
The term xe2x80x9clower alkylxe2x80x9d used in the present description denotes straight-chain or branched saturated hydrocarbon residues with 1-7 carbon atoms, preferably with 1-4 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl and the like.
The term xe2x80x9clower alkynylxe2x80x9d used in the present description denotes straight-chain or branched unsaturated hydrocarbon residues with 2-7 carbon atoms, preferably with 2-4 carbon atoms, such as ethynyl, n-propynyl, and the like.
The term xe2x80x9clower cycloalkylxe2x80x9d used in the present description denotes cyclic saturated hydrocarbon residues with 3-5 carbon atoms, preferably with 3 carbon atoms, such as cyclopropyl.
The term xe2x80x9clower alkoxyxe2x80x9d denotes a lower alkyl residue in the sense of the foregoing definition bonded via an oxygen atom.
The term xe2x80x9chalogenxe2x80x9d embraces fluorine, chlorine, bromine and iodine.
The expression xe2x80x9c5 or 6 membered heterocyclic ringxe2x80x9d embraces thiophene, furane, thiazole, pyridine, partially hydrated pyridine, for example 2-pyridone, partially hydrogenated prydine, for example tetrahydropyridine, five-membered aromatic heterocycle containing up to 4 heteroatoms, selected from O, S, N, embracing imidazol-1-yl, imidazol-2-yl, imidazol-4-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl; 1,3-thiazol-2-yl, 1,3-thiazol-4-yl, 1,3-thiazol-5-yl; 1,3-oxazol-2-yl, 1,3-oxazol-4-yl, 1,3-oxazol-5-yl, 1,2-oxazol-3-yl, 1,2-oxazol-4-yl, 1,2-oxazol-5-yl; 1,2,3-triazol-1-yl, 1,2,3-triazol-4-yl, 1,2,4-triazol-1-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-2-yl; 1,2,4-oxadiazol-2-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,3-oxadiazol-4-yl, 1,2,3-oxadiazol-5-yl; 1,2,4-thiadiazol-2-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,3-thiadiazol-4-yl, 1,2,3-thiadiazol-5-yl; tetrazol-1-yl, tetrazol-2-yl, tetrazol-5-yl;
The compounds of formula I and their pharmaceutically acceptable salts can be manufactured according to the following methods: 
Compounds of formula I, in which R1, R3 and X are as described above can be prepared according to scheme A, by, for example, cleaving the BOC protecting group in compounds of formula II, and concomitant cyclization of the deprotected compound. The deprotection-cyclization step can be carried out by treating the compounds of formula II with a bronsted acid, like for example trifluoroacetic acid (TFA), in an inert solvent, like for example dichloromethane (DCM). The reaction is preferably carried out at temperatures between 0xc2x0 C. and 50xc2x0 C. It may be advantageous to use also anisole or 1,3-dimethoxybenzene as a carbocation scavenger in the reaction mixture. Any other suitable amino protecting group, such as e.g. Fmoc or benzyloxycarbonyl (Z), can be alternatively used instead of the BOC group. 
Compounds of formula II, in which R1, R3 and X are as described above, can be prepared according to scheme B by for example reacting a compound of formula III with a dioxinone (formula IV) in an inert solvent like for example toluene or xylene at elevated temperatures, preferably between 80xc2x0 C. and 160xc2x0 C.
Alternatively, compounds of formula II can also be prepared by for example reaction of a compound of formula III with a xcex2-ketoester (formula IVa), in which R3 is as described above using the same conditions as described for the reaction with the dioxinones. 
According to scheme C, compounds of formula III in which R1 is as described above for compounds where X is a single bond can be prepared by different routes from the iodo-compound IX, depending on the nature of R1. As shown in scheme C, the key steps are coupling reactions of Suzuki- and Stille-type in presence or absence of carbonmonoxide. The exact conditions for the respective compounds can be found in the experimental part. 
According to scheme D, the key intermediate iodide IX can be prepared from commercially available 2-nitroaniline by a standard iodination-protection sequence. 
According to scheme E, compounds of formula IIIa in which R1 is as described above for compounds where X is an ethynediyl group can be prepared by different routes from the iodo-compound IX, depending on the nature of R1. As shown in Scheme E, the transformation can for example be carried out
a) by directly attaching the R1-alkynediyl-substituent via a Sonogashira-type coupling followed by the reduction of the nitro group or
b) by two stepwise Sonogashira-type couplings, in which first trimethylsilyl-acetylene is coupled to iodide IX to yield, after deprotection with sodium hydroxide in methanol, the intermediate XII which then can be transformed via a second Sonogashira-type coupling with the appropriate reactant R1xe2x80x94I, R1xe2x80x94Br or R1xe2x80x94OSO2CF3 and reduction of the nitro group to the desired compounds.
The exact conditions for the respective compounds can be found in the experimental part. 
According to Scheme F, the dioxinones and xcex2-keto esters building blocks with the formula IV and IVa can be prepared by methods known to someone skilled in the art from the corresponding carboxylic acid derivatives R3xe2x80x94COR, i.e. free acids, methyl or ethyl esters and acid chlorides. The exact conditions for the corresponding compounds can be found in the experimental part.
Another synthetic route to prepare compounds of formula Ic, in which R1 and X have the meaning as described above and R3 is a phenyl-carboxamide of formula C(O)NR4R5, in which R4 and R5 are hydrogen, lower alkyl or R4 and R5 together form a morpholino-residue or a N-methyl-piperazine is outlined in scheme G: 
The exact conditions for the respective compounds can be found in the experimental part.
Still another way to prepare compounds of formula I is the reaction of 4-aryl-8-iodo-1,3-dihydro-benzo[b][1,4]diazepin-2-ones (Formula Id, Synthetic Scheme H) with alkynes of formula R1xe2x80x94Cxe2x89xa1Cxe2x80x94, in which R1 has the meaning as described above, in a Sonogashira-coupling. 
The exact conditions for the respective compounds can be found in the experimental part.
The pharmaceutically acceptable salts can be manufactured readily according to methods known per se and taking into consideration the nature of the compound to be converted into a salt. Inorganic or organic acids such as, for example, hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid or citric acid, formic acid, fumaric acid, maleic acid, acetic acid, succinic acid, tartaric acid, methanesulphonic acid, p-toluenesulphonic acid and the like are suitable for the formation of pharmaceutically acceptable salts of basic compounds of formula I.
The compounds of formula I and their pharmaceutically acceptable salts are metabotropic glutamate receptor antagonists and can be used for the treatment or prevention of acute and/or chronic neurological disorders, such as psychosis, schizophrenia, Alzheimer""s disease, cognitive disorders and memory deficits. Other treatable indications are restricted brain function caused by bypass operations or transplants, poor blood supply to the brain, spinal cord injuries, head injuries, hypoxia caused by pregnancy, cardiac arrest and hypoglycaemia. Further treatable indications are acute and chronic pain, Huntington""s chorea, ALS, dementia caused by AIDS, eye injuries, retinopathy, idiopathic parkinsonism or parkinsonism caused by medicaments as well as conditions which lead to glutamate-deficient functions, such as e.g. muscle spasms, convulsions, migraine, urinary incontinence, nicotine addiction, psychoses, opiate addiction, anxiety, vomiting, dyskinesia and depression.
The compounds of formula I and pharmaceutically acceptable salts thereof can be used as medicaments, e.g. in the form of pharmaceutical preparations. The pharmaceutical preparations can be administered orally, e.g. in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. However, the administration can also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions.
The compounds of formula I and pharmaceutically acceptable salts thereof can be processed with pharmaceutically inert, inorganic or organic carriers for the production of pharmaceutical preparations. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts and the like can be used, for example, as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like; depending on the nature of the active substance no carriers are, however, usually required in the case of soft gelatin capsules. Suitable carriers for the production of solutions and syrups are, for example, water, polyols, sucrose, invert sugar, glucose and the like. Adjuvants, such as alcohols, polyols, glycerol, vegetable oils and the like, can be used for aqueous injection solutions of water-soluble salts of compounds of formula I, but as a rule are not necessary. Suitable carriers for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.
In addition, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
As mentioned earlier, medicaments containing one or more compounds of formula I or a pharmaceutically acceptable salt thereof and a therapeutically inert excipient are also an object of the present invention, as is a process for the production of such medicaments which comprises bringing one or more compounds of formula I or pharmaceutically acceptable salts thereof and, if desired, one or more other therapeutically valuable substances into a galenical dosage form together with one or more therapeutically inert carriers.
The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, the effective dosage for oral or parenteral administration is between 0.01-20 mg/kg/day, with a dosage of 0.1-10 mg/kg/day being preferred for all of the indications described. The daily dosage for an adult human being weighing 70 kg accordingly lies between 0.7-1400 mg per day, preferably between 7 and 700 mg per day.
The present invention relates also to the use of compounds of formula I and of pharmaceutically acceptable salts thereof for the production of medicaments, especially for the control or prevention of acute and/or chronic neurological disorders of the aforementioned kind.
The compounds of the present invention are group II mGlu receptor antagonists as determined using the assay described by Cartmell et al. (Br. J. Pharmacol. 1998, 123(3), 497-504).
The compounds show activities, as measured in the assay described below, of 50 xcexcM or less, typically 3 xcexcM or less, and ideally of 0.5 xcexcM or less. In the table below are described some specific pKi values of preferred compounds
Transfection and Cell Culture
cDNA encoding the rat mGlu2 receptor protein in pBluescript II was obtained from Prof. S. Nakanishi (Kyoto, Japan), and subcloned into the eukaryotic expression vector pcDNA I-amp from Invitrogen (N V Leek, The Netherlands). This vector construct (pcD1mGR2) was co-transfected with a psvNeo plasmid encoding the gene for neomycin resistance, into CHO cells by a modified calcium phosphate method described by Chen and Okayama (1988). The cells were maintained in Dulbecco""s Modified Eagle medium with reduced L-glutamine (2 mM final concentration) and 10% dialysed foetal calf serum from Gibco BRL (Basel, Switzerland). Selection was made in the presence of C-418 (1000 ug/ml final). Clones were identified by reverse transcription of 5 xcexcg total RNA, followed by PCR using mGlu2 receptor specific primers 5xe2x80x2-atcactgcttgggtttctggcactg-3xe2x80x2 and 5xe2x80x2-agcatcactgtgggtggcataggagc-3xe2x80x2 in 60 mM Tris HCl (pH 10), 15 mM (NH4)2SO4, 2 mM MgCl2, 25 units/ml Taq Polymerase with 30 cycles annealing at 60xc2x0 C. for 1 min., extension at 72xc2x0 C. for 30s, and 1 min. 95xc2x0 C. denaturation.
Membrane Preparation
Cells, cultured as above, were harvested and washed three times with cold PBS and frozen at xe2x88x9280xc2x0 C. The pellet was resuspended in cold 20 mM HEPES-NaOH buffer containing 10 mM EDTA (pH 7.4), and homogenised with a polytron (Kinematica, AG, Littau, Switzerland) for 10s at 10,000 rpm. After centrifugation for 30 min. at 4xc2x0 C., the pellet was washed once with the same buffer, and once with cold 20 mM HEPES-NaOH buffer containing 0.1 mM EDTA, (pH 7.4). Protein content was measured using the Pierce method (Socochim, Lausanne, Switzerland) using bovine serum albumin as standard.
[3H]-LY354740 Binding
After thawing, the membranes were resuspended in cold 50 mM Tris-HCl buffer containing 2 mM MgCl2 and 2 mM CaCl2, (pH 7) (binding buffer). The final concentration of the membranes in the assays was 25 xcexcg protein/ml. Inhibition experiments were performed with membranes incubated with 10 nM [3H]-LY354740 at room temperature, for 1 hour, in presence of various concentrations of the compound to be tested. Following the incubations, membranes were filtered onto Whatmann GF/C glass fiber filters and washed 5 times with cold binding buffer. Non specific binding was measured in the presence of 10 xcexcM DCG IV (TOCRIS No. 0975). After transfer of the filters into plastic vials containing 10 ml of Ultima-gold scintillation fluid (Packard, Zxc3xcrich, Switzerland), the radioactivity was measured by liquid scintillation in a Tri-Carb 2500 TR counter (Packard, Zxc3xcrich, Switzerland).
Data Analysis.
The inhibition curves were fitted with a four parameter logistic equation giving IC50 values, and Hill coefficients.